Electric fields have been employed in several different types of cancer therapy. Some of these involve radio frequency or microwave devices that heat the tumor to greater than 43° C. to kill the cells via hyperthermia (K. K. Tanabe, S. A. Curley, G. D. Dodd, A. E. Siperstein, S. N. Goldberg (2004) Cancer. 100:641-650; D. Haemmerich, P. F. Laeseke (2005) Int. J. Hyperthermia. 21:755-760). Others use pulsed electric fields to permeabilize the tumor cells to allow the introduction of toxic drugs or DNA (M. L. Lucas, R. Heller (2003) DNA Cell Biol. 22:755-763; Y. Kubota, Y. Tomita, M. Tsukigi, H. Kurachi, T. Motoyama, L. M. Mir (2005) Melanoma Res. 15:133-134; A. Gothelf, L. M. Mir, J. Gehl (2003) Cancer Treat. Rev. 29:371-387). Previous studies have shown that fibrosarcoma tumors, treated in situ with nanosecond pulsed electric fields, exhibited a reduced growth rate compared to control tumors in the same animal (S. J. Beebe, P. Fox, L. J. Rec, K. Somers, R. H. Stark, K. H. Schoenbach (2002) IEEE Transactions on Plasma Science. 30:286-292).
The main characteristics of nanosecond pulsed electric fields (nsPEF) are their low energy that leads to very little heat production and their ability to penetrate into the cell to permeabilize intracellular organelles (K. H. Schoenbach, S. J. Beebe, E. S. Buescher (2001) Bioelectromagnetics. 22:440-448; E. S. Buescher, K. H. Schoenbach (2003) IEEE Transactions on Dielectrics and Electrical Insulation. 10:788-794) and release calcium (P. T. Vernier, Y. H. Sun, L. Marcu, S. Salemi, C. M. Craft, M. A. Gundersen (2003) B B R C. 310:286-295; E. S. Buescher, R. R. Smith, K. H. Schoenbach (2004) IEEE Transactions on Plasma Science 32:1563-1572; J. A. White, P. F. Blackmore, K. H. Schoenbach, S. J. Beebe (2004) J. Biol. Chem. 279:22964-22972) from the endoplasmic reticulum (J. A. White et al. (2004) J. Biol. Chem). They provide a new approach for physically targeting intracellular organelles with many applications, including the initiation of apoptosis in cultured cells (S. J. Beebe, P. M. Fox, L. J. Rec, E. L. Willis, K. H. Schoenbach (2003) FASEB J. 17:1493-1495; S. J. Beebe, J. White, P. F. Blackmore, Y. Deng, K. Somers, K. H. Schoenbach (2003) DNA Cell Biol. 22:785-796; S. J. Beebe, P. F. Blackmore, J. White, R. P. Joshi, K. H. Schoenbach (2004) Physiol Meas. 25:1077-1093) and tumors (S. J. Beebe et al. (2002) IEEE Transactions on Plasma Science) enhancement of gene transfection efficiency (S. J. Beebe et al. (2003) DNA Cell Biol; S. J. Beebe et al. (2004) Physiol Meas.) and reducing tumor growth (S. J. Beebe et al. (2002) IEEE Transactions on Plasma Science).
The use of electric fields on biological cells to rupture the cell membrane can lead to cell death via necrosis, a nonphysiological type of cell destruction, while the use of nsPEFs on biological cells to permeabilize intracellular organelles can initiate cell death via apoptosis. When treating biological cells within tissue in situ, being able to initiate cell death via apoptosis would allow the destruction of specific undesired cells in situ without engendering the non-specific damage to surrounding or nearby tissue in the body due to inflammation and scarring that is normally observed with necrosis. Investigations of the effects of ultrashort, high intensity pulsed electric fields or nanosecond pulsed electric fields (nsPEF) on mammalian cells have demonstrated distinct differences on cell structure and function compared to classical plasma membrane electroporation. It was previously demonstrated that nsPEF invoked signal transduction mechanisms that initiate apoptosis cascades in several human cell lines including HL-60 cells (Beebe, S. J., et al. (2002) IEEE Trans. Plasma Sci. 30, 286-292; Beebe, S. J., et al. (2003) FASEB J. 17, 1493-1495).
The efficacy of this nsPEF treatment is believed to depend on two separate electric field parameters: pulse duration and amplitude. The effect of pulse duration can be understood by considering the process of membrane charging when the cell is placed in an electric field. Ions in the cell interior will respond to the electric field by moving in the field direction and charging the highly resistive membrane until they experience no further force. By definition this will only occur when their redistribution establishes an equal and opposite field so that the net electric field in the cell interior is zero. However this redistribution takes a certain amount of time that is characterized by the charging time constant of the plasma membrane, typically in the 0.1 to 1 microsecond range. If the nsPEF is shorter than this charging time, the interior charges will not have sufficient time to redistribute to counteract the imposed field and it will penetrate into the cell and charge every organelle membrane for a duration which is dependent on both the charging time constant of the cell's plasma membrane as well as that of the organelle membrane (K. H. Schoenbach, R. P. Joshi, J. F. Kolb, N. Chen, M. Stacey, P. F. Blackmore, E. S. Buescher, S. J. Beebe (2004) Proc. IEEE. 92:1122-1137).
A second critical nsPEF parameter is the amplitude of the pulse. Both the force exerted on charges and the electroporation of lipid membranes depend on the strength of the electric field. When the electric field across a cellular membrane exceeds about 1 volt (2 kV/cm for a cell 10 μm in diameter), water-filled pores form in the membrane's lipid bilayer and the size and lifetime of these pores are dependent on the strength and duration of the electric field pulse. For amplitudes exceeding 2 kV/cm and pulse durations in the millisecond range, large pores form resulting in electroporation of the membrane that has been used to introduce normally impermeant anticancer drugs into targeted tissues (M. L. Lucas et al (2003) DNA Cell Biol.; Y. Kubota et al (2005) Melanoma Res.; A. Gothelf et al (2003) Cancer Treat. Rev.; J. Teissie, M. Golzio, M. P. Rols (2005) Biochim. Biophys. Acta 1724:270-280). For these long pulses, the pulse amplitude is limited to about 2 kV/cm to avoid thermal effects. Since heating is proportional to pulse duration and the square of the field strength, the much shorter pulses in the nanosecond range can have a higher field strength while delivering the same low level of thermal energy to the tissue. A 20-fold higher field strength of 40 kV/cm can be employed to generate structural changes in the plasma membrane that result in a smaller electrical barrier as well as higher voltage gradients across cellular organelles for the duration of the pulse (Q. Hu, S. Viswanadham, R. P. Joshi, K. H. Schoenbach, S. J. Beebe, P. F. Blackmore (2005) Phys. Rev. E Stat. Nonlin. Soft. Matter Phys. 71:031914-1-031914-9). A typical tumor cell nucleus measuring 10 μm in diameter will experience a voltage gradient of roughly 40 V across its diameter during each pulse. This electric field is large enough to cause electrodeformation (R. P. Joshi, Q. Hu, K. H. Schoenbach, H. P. Hjalmarson (2002) Phys. Rev. E Stat. Nonlin. Soft. Matter Phys. 65:021913).
Previous studies provided direct evidence for cellular DNA as a direct or indirect target of nsPEF. Using a comet assay, Stacey, et al. (M. Stacey, J. Stickley, P. Fox, V. Statler, K. Schoenbach, S. J. Beebe, S. Buescher (2003) Mutat. Res. 542:65-75) found that ten 60 ns pulses of 60 kV/cm caused a rapid 2.6-fold increase in the mean image length of DNA electrophoresis tracks in Jurkat cell extracts and a 1.6-fold increase in the comet assay from HL60 cell extracts. In both cases this was a very significant change (p<0.001). This elongation in DNA electrophoresis tracks is normally interpreted to indicate fragmentation of the DNA into smaller pieces that is associated with apoptotic cell death. An indication of changes in the DNA following nsPEF treatment comes from images of the nucleus labeled with acridine orange, a vital fluorescent dye that intercalates into DNA and RNA, Chen et al. (N. Chen, K. H. Schoenbach, J. F. Kolb, S. R. James, A. L. Garner, J. Yang, R. P. Joshi, S. J. Beebe (2004) Biochem. Biophys. Res. Commun. 317:421-427). A single 10 ns pulse of 26 kV/cm caused a dramatic decrease in fluorescence intensity in the nucleus evident as early as 5 min after the pulse. This change could be due to an outflow of DNA or to conformational changes in the DNA.
The ability to selectively modify specific cells in ways that lead to apoptosis could provide a new method for the selective destruction of undesired tissue (e.g., cancer cells, fat cells or cartilage cells) while minimizing side effects on surrounding tissue. An electrical method of treatment that results, not only in tumor growth inhibition, but in complete tumor regression, without hyperthermia, drugs, or significant side effects, would be a great advancement in the field of cancer therapy and other in situ therapies. These and various other needs are addressed, at least in part, by one or more embodiments of the present invention.